Currently, the olive oil extraction system according to a two-phase extraction process [J. Alba, F. Hidalgo, Ma A Ruiz, F. Martínez, Ma J. Moyano, A. Cert, Ma C. Pérez and Ma V Ruiz. “Características de los aceites de oliva de primera y segunda centrifugación” Grasas y Aceites 47, (1996) 163–181] has substantially reduced the volume of wastes produced with the traditional three-phase system which requires the supplementary addition of water. Nevertheless, there is still a high quantity (approximately 2,600,000 tons/year in Spain) produced of a residue, called two-phase pomace or “alpeorujo”, formed by the pulp, the pit and the vegetable water. The depleted product, with a fat content lower than 1% and a humidity around 65%, is an inconvenient and very abundant residue (around 80% of the ground olive), impossible to store in the olive-oil mill. Due to the environmental, economic and social problems caused by the large quantity of sub-product generated, it is necessary to find solutions for a comprehensive treatment thereof, which is feasible from an economic point of view and does not negatively affect the environment.
The reuse of this sub-product in the conventional olive-pomace oil extractors for an additional extraction of the remaining oil contained therein, poses a difficult problem associated with its characteristics of having a paste consistency and a high content in sugars and fine solids, which make its use as such very difficult in conventional olive-pomace drying systems. Although several investigations are being performed to optimize the drying process from an operational and energy-saving point of view [R. Arjona, A. García and P. Ollero. “The drying of alpeorujo, a waste product of the olive mill industry”, Journal of Food Engineering 41 (1999) 229–234], the high energy cost of the humidity reduction phase is a clear drawback from the economic point of view. Another alternative treatment to reduce the volume of this residue, such as its incineration in combustion plants, also requires the prior drying of the material and may also imply a series of environmental problems related to the emission of toxic exhaust gases. In this regard, it would be necessary to use suitable gas cleaning systems, which would substantially increase the overall economic cost of the process.
The alpeorujo contains all the sugars and other soluble substances that are dissolved in the vegetable water of the conventional olive oil extraction process. Thus, there is a possibility of revaluating this residue by the production of products of high added value therefrom, which would mean an improvement in the management of sub-products from the olive-producing industry, whilst permitting the use of a cheap source of raw materials for products of industrial interest. An example of this approximation to the revaluation of alpeorujo is in patent application ES 2060 549, which discloses a process to produce mannitol and products derived from alpeorujo, by processes of diluting, filtration, decanting or centrifuging.
In addition to compounds such as mannitol, the majority of the polyphenols present in the olive pulp and pit are found in this sub-product, for which reason it may constitute a suitable source of natural phenolic antioxidants. The phenolic compounds of the olive pulp have been extensively researched given their importance in fruit processing. The most abundant glycoside in olive pulp, oleuropein, is formed by the heterosidic ester of elenolic acid and hydroxytyrosol and is what gives olives their bitter taste. Oleuropein is very abundant in the green fruit and their percentage is quantitatively reduced on reaching maturity. Ligustroside and verbascoside are also present in the pulp. In the pit, the described glycosides are similar to the above and of the ligustroside type, of which tyrosol instead of hydroxytyrosol forms a part.
The most abundant phenolic compounds in alpeorujo are tyrosol and hydroxytyrosol, together with p-coumaric, caffeic, ferulic and vanillic acids in less quantity. The hydroxytyrosol mainly comes from the oleuropein of the fruit, which is hydrolysed by means of an esterase present therein during the olive grinding. These polyphenols, which are mainly present in the fruit, but also in less quantity in the leaves, flowers and other plant organs, are natural antioxidant compounds, which perform various functions. In a recent study, their antioxidant effect has been proven as they block the process in the initiation phase, either oxidizing themselves by forming more stable natural peroxidic compounds, or acting on the already formed radicals to prevent the propagation phase. On the one hand, the phenols present in the olive protect the oil from degrading [Gutfinger, T. “Polyphenols in olive oils” J. Am. Oil Chem Soc. 58 (1981) 966–7], and they have infinite potent biological actions on the human metabolism, which have been studied in vitro. The most important of them is the inhibition of low-density lipoprotein (LDL) oxidation, which constitutes an essential phase within the atherogenic process. However, phenolic compounds also have important free-radical scavenging activity [F. Visioli, G. Bellomo, C. Galli. “Free radical-scavenging properties of olive oil polyphenols”. Biochem Biophys Res. Municipality 247 (1998) 60–64] against the production of the superoxide anion and hypochlorous acid, which means they act as protection against oxygen free radicals and their toxic effects and, therefore, prevent cellular aging and tumours [R. W Owen, A. Giacosa, W. E Hull, R. Haubner, B. Spiegelhalder, H. Bartsch. “The antioxidant/anticancer potential of phenolic compounds isolated from olive oil” Eur. J. Cancer 36(10), (2000) 1235–1247], they protect against heart disease and are closely related to the benefits attributable to the consumption of olive oil in the Mediterranean diet. Some of them, such as hydroxytyrosol, are potent inhibitors of platelet aggregation and thromboxane generation and counteract the production of the potent pro-inflammatory agent leukotriene B4.
The high potential for these phenolic compounds in the pharmaceutical and food industry has caused a growing interest in the last few years in their production from olive leaves and fruit for various applications. For example, the Japanese patent application JP08119825 discloses the application of the hydroxytyrosol obtained from olive oil extraction as an active component in cutaneous formulations for skin treatments. The patent application WO 00/36936 discloses the preparation of food compositions which have increased levels of phenolic compounds from olive oil to improve their nutritional qualities. The patent U.S. Pat. No. 6,165,475 relates to the use of oleuropein in various medical or agricultural applications, as well as a method to prepare an aqueous extract rich in this compound which comes from the fruit. Nevertheless, there are almost no references on the use of plant materials, such as the pulp, that remain after the extraction of the untreated olive oil (crude), as a substrate for the recovery of polyphenols, the use of this crude substrate forming one of the novel aspects of the present invention. A work published by the applicants for the production of bioethanol from alpeorujo [I. Ballesteros, J. M Oliva, M. J. Negro, P. Manzanares, M. Ballesteros. “Ethanol production from olive oil extraction residue pretreated with hot water” Applied Biochemistry and Biotechnology, 98–100 (2002) 717–732], discloses that, after a hydrothermal treatment performed to promote the subsequent transformation of the cellulose contained in the substrate to ethanol, phenolic compounds are obtained such as those disclosed in the present application. However, the residue used in said work has undergone a washing and drying process before being used with the aim of promoting the subsequent fermentation process to which it is subjected. Surprisingly, the applicants have now found that the use of this substrate when crude, i.e. as it is received from the olive-mill, without undergoing any prior washing and drying treatment, is advantageous in a process to extract phenolic compounds as is disclosed in this application. Firstly, the use of crude alpeorujo avoids the expense of water in the washing phase and the subsequent generation of a liquid residue, whose management may be problematic from an environmental point of view. Furthermore, it is a clear advantage from an energy-saving point of view as it avoids the drying phase, after that of the washing, which is disclosed in the aforementioned work. Finally, as it uses the crude substrate, a higher yield of hydroxytyrosol and tyrosol are obtained than that corresponding to the same substrate after being washed and dried.
As regards the extraction methods of the phenolic compounds mainly present in the leaves and the fruit, these vary depending on the type of starting material. Thus, polyphenol extraction methods with organic solvents such as methanol/water have been disclosed to extract from fruit [D. Ryan, H. Lawrence, P. Prenzier, M. Antolovich, K. Robards. “Recovery of phenolic compounds from Olea europaea” Analytica Chimica Acta 445 (2001), 67–77], alcohol to extract from leaves (patent U.S. Pat. No. 5,714,150) and ether/ethyl acetate [A. D. Bianco, I. Muzzalupo, A. Piperno, G. Romeo, N. Uccella. “Bioactive derivatives of oleuropein from olive fruits” J. Agric. Food Chem. 47 (1999), 3531–3534] or methanol [F. Angeroso, N. D'Alessandro, Konstantinou. P., L. Di Giacinto. “GL-LC evaluation of phenolic compounds in virgin olive oil” J. Agric. Food. Chem. 43 (1995), 1802–1807] applied to polyphenol extraction from olive oil L. Lesage-Meessen et al., [L. Lesage-Meessen, D. Navarro, S. Maunier, J-C Sigoillot, J. Lorquin, M. Delattre. J-L Simon, M. Asther and M. Labat. “Simple phenolic content in olive oil residues as a function of extraction systems” Food Chemistry 75(4) (2001) 501–507, disclose the extraction of tyrosol and hydroxytyrosol from the residue obtained from olive oil using ethyl acetate, permitting the recovery of around 30% of the total amount of phenols present in the residue. However, all these methods have the drawback of their high cost as regards the use of solvents and the generation of wastes which are difficult to eliminate.
The patent WO 01/45514 discloses a method to extract antioxidant compositions from the fruit and olive oil extraction sub-products, in a multi-phase process that uses several aqueous and/or polar extractions and solid matrixes which is considerably more complex than the present application. The Spanish patent application ES 2143939 discloses the use of a steam explosion process to recover the structural phenolic components from the olive pits and the kernel shell. With this process, the materials are treated in a 2 l steam explosion unit at temperatures around 200° C. for time periods of 2–4 minutes, there then occurring an abrupt decompression and the subsequent unloading of the reactor. The results obtained show that hydroxytyrosol is extracted from the pit in soluble extract concentrations of up to 1% in dry weight of the pit, less quantity than that obtained in the pulp, where this polyphenol is mainly found. In the case of the kernel shells, tyrosol is obtained in concentrations of up to 0.5% in dry weight and it is verified that the addition of acid to the material before the treatment appreciably increases the quantities of phenols detected in the soluble extract.
The object of the present invention is to extract the phenolic derivatives present in crude residual plant materials, in particular, in the residue remaining after olive oil extraction by a two-phase centrifugation process (alpeorujo), using a hydrothermal treatment that uses water at a high temperature and at a pressure to keep the water in liquid phase.
In the sense used in this description, the term “crude residual plant materials” includes residues of plant origin which contain phenolic derivatives, in particular phenolic derivatives of industrial interest, such as alpeorujo, olive pit debris, olive kernel shell, corncob residues, etc, which have not undergone, prior to their use, any washing and/or drying process.
As is used in this description, phenolic derivatives of industrial interest include the phenols tyrosol and hydroxytyrosol.
In the present invention, a hydrothermal treatment is used, which uses hot water in liquid phase, to extract phenolic derivatives from the crude olive oil extraction residue. This application of treatment with hot water to the recovery of phenolic compounds, is a new use thereof which, up to the present time, has basically been used as a pre-treatment of lignocellulosic materials prior to an enzymatic hydrolysis of the cellulose to glucose, as disclosed in the patent U.S. Pat. No. 5,846,787. This treatment, in the case of crude alpeorujo, is an advantage to the aforementioned extraction methods as it allows the recovery of phenols of interest without the exogenous addition of solvents or acids, which means the overall process is more economic and gives less quantity of residues to eliminate.
The method of extraction by hydrothermal treatment of this invention is based on subjecting the material to be treated to a treatment with hot water, at a temperature of 180 to 240° C., in an closed autoclave-type reactor, at a pressure that permits maintaining the water in liquid phase, during an appropriate time period, of 4 to 30 minutes, with the aim of solubilizing the phenolic derivatives, mainly present in said material. Next, the liquid fraction is recovered by filtration and its phenol concentration is determined. During the treatment of the substrate of the present invention, an autohydrolysis of the hemicellulosic debris occurs in said pulp residue that produces the liberation of acetyl groups and, in consequence, a reduction in the pH of the liquid fraction, thus promoting the solubilization of the phenols of interest. Once the phenolic derivatives have been extracted, their concentration in the aqueous extracts is determined by high performance liquid chromatography (HPLC) techniques or gas chromatography-mass spectometry (GC-MS) or spectroscopy techniques, which are well known by those skilled in the art.